Antithrombotic derivatives of ammonium ascorbate

ABSTRACT

A solution of L-ascorbic acid and a solution of para amino benzoic acid are mixed and the mixture evaporated to permit recovery of a solid compound. The compound is administered to a host to reduce thrombotic tendencies.

OTHER APPLICATIONS

This application is a continuation-in-part of our earlier filedapplication Ser. No. 35,637, filed May 3, 1979 now abandoned, which is acontinuation of application Ser. No. 701,874 filed July 1, 1976 (nowU.S. Pat. No. 4,164,585).

FIELD OF INVENTION

This invention relates to compounds of organic acids and to methods ofmaking and using the same particularly for pharmaceutical purposes.

BACKGROUND OF THE INVENTION

U.S. Pat. No. 3,722,504 describes a screen for testing pharmaceuticalcompounds based at least partly on their ability to increase thenegative surface charge of the vascular system. U.S. Pat. No. 3,722,504,by way of example, discloses a pharmaceutical compound which preventsthrombosis by modifying the intimal surface charge of the vascularsystem.

Para amino benzoic acid increases the net negative surface charge ofblood vessels and blood cells. It produces marked increases in thecurrent induced occlusion time in rat mesentary vessels (see U.S. Pat.No. 3,956,504). It produces no measurable effect on blood coagulationstudies. It has a limited effect on blood vessel wall pores as shown byelectro-osmotic studies.

Para amino benzoic acid, further, has good antithromboticcharacteristics but tends to have relative little anti-coagulantactivity as shown by its lack of effect on the coagulation studiesincluding partial thromboplastin time, thrombin time and recalcificationtime. It is therefore a very useful antithrombotic drug.

Para amino benzoic acid has various very significant properties as adrug:

1. It is inexpensive;

2. It is known to be non-toxic in humans;

3. It can be used for long periods of time without significant incidenceof pathological manifestations.

4. The material can be taken orally in large dosages without significanttoxicity.

In man, dosages have been administered, for example, as high as 12 to 24grams a day without significant side effects. It has furthermore beenused for a large number of diseases in man including the therapy ofRicketsial diseases before the development of anti-biotics, fortuberculosis in very large dosage and in the treatment of certainprotozoan diseases and other infestation. It has long been thought to bea mild anti-inflammatory agent.

In humans with arteriosclerotic peripheral vascular disease, para aminobenzoic acid has been shown to increase blood flow in ischemic limbs asmeasured by Barcroft plethysmographic studies and Doppler blood flowmeasurements. Studies which were carried out in approximately fiftypatients resulted in an approximate doubling of blood flows in patientsin which para amino benzoic acid salt was effective. U.S. Pat. No.3,956,504 has been issued on the effect of para amino benzoic acid onblood flow in man.

The long term effects of vitamin C on scurvy in man is a primary eventof historical interest in medicine. The effects of vitamin C to preventscurvy are extraordinarily well documented throughout the world. Man,deprived of vitamin C for periods longer than approximately sixty days,starts to display evidence of capillary fragility and bleeding into alltissues and organs. Vitamin C has the following characteristics:

1. It is a co-enzyme in the Kreb's cycle.

2. It is a reducing agent and electron donor in all known biologicalsystems.

L-ascorbic acid or vitamin C has a variety of biological functions someof which are not completely understood (Zitent et al. 1964). Large dosesof L-ascorbic acid appear to have value in prevention and symptomreduction in the common cold and other viral diseases (Linus Pauling1974). Recent experimental evidence indicates that L-ascorbic acid iseffective in reducing serum cholesterol levels. C. Spittle (The Lancet,July 28, 1973; pp. 199-201 and Dec. 11, 1971; pp. 1280-1281) suggestedthat dosages of 1 to 2 grams of vitamin C per day can be beneficial inthe prevention of deep vein thrombosis as well as in reducing theincidence of atherosclerotic complications in man. Recent researchindicates that ascorbic acid may reduce the incidence of myocardialinfarction (Knox, E. G., The Lancet, pp. 1465, June 30, 1973).L-ascorbic acid, further, appears to offset the thrombogenic effects oforal contraceptives as demonstrated by prolongation of occlusion timesin the mesenteric vessels.

Good results were obtained in a pilot study using seven volunteersubjects to determine the effects of ascorbic acid on (1) plasmacoagulation characteristics (2) serum cholesterol levels (3) plateletaggregability and (4) surface charge characteristics of red cells andplatelets. Vitamin C in these subjects was shown to decrease plateletaggregability and increase the electro-phoretic mobility of all thetested cells.

There has been little evidence to indicate that vitamin C effectsmeasured blood coagulability as demonstrated by partial thromboplastintime, thrombin times and thrombin recalcification times.

Spittle et al. have tested vitamin C in a randomized group of patientswith thrombophlebitis. The protective effect of vitamin C againstthrombosis has been shown by a randomized double-blind trial usingpatients who were shown to be prone to deep-venous thrombosis. In atotal of fifty-three patients, it was observed that the incidence ofdeep venous thrombosis was 33% in patients dosed with L-ascorbic acidcompared to 60% in the placebo group (Spittle, C. R., The Lancet, pp.199, July 28, 1973). The dose given was 1 gram per day. This correlationbetween the intake of vitamin C and reduction in the number ofthrombotic episodes has been confirmed by other sources. In burnpatients, where it is customary to use large doses of vitamin C to speedhealing, there has been an apparent demand for treatment of deep-veinthrombosis. This information is based on the experience of one hospitalover a five and one-half year period during which time 159 patients overforty years of age were treated with large doses of vitamin C (Spittle,C. R. "The Action of Vitamin C on Blood Vessels," Amer. Heart J. 88:387,1974).

A combination of para amino benzoic acid and vitamin C has been shown,for example, in Runti, Il Farmaco, Ed. Sci., Vol, X, 1955, pp. 424-431.

SUMMARY OF THE INVENTION

It is an object of the invention to provide an improved combination ofpara amino benzoic acid and L-ascorbic acid.

It is another object of the invention to provide an improvedpharmaceutical compound having improved utility for extended periodsfollowing administration.

Yet another object of the invention is to provide improved methods forthe treatment of hosts and the prevention of vascular conditions.

Still another object is to promote the development of useful compoundsbased on organic acids and the like.

A further object of the invention is to provide new methods for thedevelopment of pharmaceutical compounds.

To achieve the above and other objects of the invention, there isprovided a method comprising reducing thrombotic tendencies in a host byadministering to the host a compound derived from two organic acids.

According to one embodiment of the invention, one of the acids isL-ascorbic acid and the other is para amino benzoic acid.

The compound which is derived may be a salt or ester and this compoundmay be administered in a dosage of 1-100 mg./kg. of body weight. Thedosage is preferably administered orally, although it can beadministered interperitoneally.

The compound may be formed by mixing solutions of L-ascorbic acid andvarious forms of para amino benzoic acid and recovering the thuslyresulting solid.

The invention includes, as one aspect thereof, compounds preparedaccording to the above method or specifically salts and esters ofL-ascorbic acid and para amino benzoic acid to obtain:

A. p-carboethoxy phenyl amino ascorbate the salt of ascorbic acid andethyl p-amino benzoate ##STR1##

B. p-glyceryl carboxy phenyl amino ascorbate the salt of ascorbic acidand glyceryl para amino benzoic acid ##STR2##

C. esters of para amino benzoic acid and ascorbic acid ##STR3##

BRIEF DESCRIPTION OF DRAWING

The sole FIGURE of the drawing is a chart demonstrating the prolongedactivity of dosages of the compound of the invention.

DETAILED DESCRIPTION

Hereinabove, reference had been made to para amino benzoic acid. Theformula for this organic acid is as follows: ##STR4##

Reference has also been made to L-ascorbic acid or vitamin C, theformula for which is: ##STR5##

The present invention relates to compounds derived from these organicacids, namely, salts, esters and amides thereof, the synthesization ofthe same and the applicability thereof to the treatment of the vascularsystem, notably in rats, dogs and human hosts requiring such treatment.

The formula for the salt of our earlier U.S. Pat. No. 4,164,585 is asfollows: ##STR6##

The original approach was to prepare a salt of ascorbic acid with paraamino benzoic acid. Such a salt can be shown in two forms--one using theopen form of ascorbic acid, the other using the enol form.

Two manufacturing procedures used are described below. Because of theease of oxidation of ascorbic acid, it is essential to perform alloperations under nitrogen and free of water and air. In one case,substantial darkening of the product resulted. It was believed this wasdue to the presence of moisture.

EXAMPLE I

1. A solution of para mino benzoic acid in a mixture of 50% acetone and50% methanol was prepared with gentle heating. The concentration wasapproximately 100 grams per liter.

2. A similar solution was prepared with L-ascorbic acid.

3. The two solutions were mixed. Water was excluded to the extentpossible.

4. The mixture was evaporated under vacuum, with nitrogen being utilizedto flush the system. As the material concentrated, crystals began toappear.

5. When the solution was evaporated, until it was a thick slurry, thematerial was filtered through a coarse filter paper and allowed to drainas dry as possible. The surface was blanketed with nitrogen, with greatcare to exclude all filtered water.

6. The resultant solid was dried under high vacuum at room temperature.The ratio of L-ascorbic acid to para amino benzoic acid used was one toone on a molar basis.

EXAMPLE II

1. An alternative method of preparation used was to evaporate thesolvent with careful exclusion of water and air to the point where asolid semi-dry cake was obtained in the container in which theevaporation was conducted.

2. This moist cake was then transferred to an appropriate container anddried under high vacuum. All operations are protected against exposureto air and moisture.

Other solvent systems will undoubtedly work. The above were used largelyas a matter of convenience. An odor develops in the process which isremoved with high vacuum drying, but must represent a by-product whichis formed during the process. It must also be volatile, since it can beremoved.

The first salt shown above, theoretically, only requires the removal ofa molecule of water to form the amide. In general, this does not happentoo readily. The distillation of the solvents may help pull off water.

Initial studies were directed toward determining the toxicity of theabove-indicated compound in mammalia.

A very high dosage per kilogram of body weight in rats and dogs has beenshown essentially non-toxic.

The new compound was next tested to determine its effect onrat-mesentery occlusion studies. Half lives of para amino benzoic acidhave been shown to approximate one day. Half lives of vitamin Capproximate 12 hours to a maximum of 1 day. The half lives of the newcompound have been shown to approximate 4 to 5 days with a tail. Asingle dose lasts approximately 14 days. Mechanical mixing of para aminobenzoic acid and L-ascorbic acid have been shown to have a maximal tailof approximately 5 to 6 days indicating by direct logic that the newcompound is biologically different from the two components mixingtogether mechanically. Specifically, the compound derived appears to bemore potent than the starting materials mixed together in a 50/50 ratio.If the agents are mixed together in the same ratio as used when makingthe new compound, the mechanical mixture is still not as potent as thecompound derived.

The new compound has been evaluated in rat-mesentery studies. In therat-mesentery, the new compound has been shown to prolong coagulation 3to 4 times the normal rat-mesentery thrombosis time. The result isdramatic and prolonged since the effect of a single dose appears toextend out to 14 days before rat-mesentery occlusion times return to anormal level. This result is based on 26 rats.

Blood cells of male dogs fed with the new compound display an increasein negative surface charge which increases sequentially from the thirdday to approximately the 14th day, so that at 14 days there is adoubling of electrophoretic mobility over the effects seen on the fifth,sixth and seventh day.

The available evidence suggests that, as with most normal pharmacologicagents, the new compound will not produce super normality. It will,however, return toward normal any grossly abnormal measurementsconcerning the electrokinetic characteristics of blood cells and bloodvessels in dogs.

The available evidence indicates that the new compound derived fromL-ascorbic acid and para amino benzoic acid is a rather potentanti-thrombotic agent. Its effect is cumulatively greater than theeffect of either of its components even when they are mechanically mixedtogether and given orally to rats and/or dogs. The compound is anelegant example of an electron donor compound which is useful intreating the vascular system.

The following are some results of tests comparing the new compound witha mixture of the starting materials and with the starting materialsindividually:

                                      TABLE I                                     __________________________________________________________________________    Dosages - Oral Route                                                                 RANGE MGS/KG/DAY                                                                           DURATION                                                  MATERIAL                                                                             Minimum                                                                              Maximum                                                                             ADMINISTRATION                                                                           TOXICITY EFFECT                                __________________________________________________________________________    PABA (X)                                                                             1      10    Indefinite Low cutaneous                                                                          Relatively                                                           manifestation.                                                                         limited                               L-Ascorbic                                                                           0.25   10    Indefinite Low cutaneous                                                                          Critical in                           Acid (Y)                                                                             mgs.                    manifestation.                                                                         maintenance                                                          Gastritis. Some                                                                        of tissue                                                            evidence dis-                                                                          integrity                                                            turbence gene                                                                          particularly                                                         pool in massive                                                                        vascular tree                                                        dosage. Catalytic                                                             oxidizer Krebs                                                                cycle essential                                                               vitamins.                                      X + Y  1      100mg/kg                                                                            Unknown    Very low gas-                                                                          Prolonged rat                         (mixture)     rats  long term  tritis in one                                                                          mesentery                                                            rat, in high                                                                           occlusion                                                            dosage 1mg/gm                                                                          time. Single                                                         body weight                                                                            dose run                                                             dosage.  (day 1):                                                                      170 ± 20 min.                                                              Tail - 7 days.                        X - Y  2      100mg/kg                                                                            May be     Very low Prolonged.                            (compound)          given long gastritis.                                                                             Longer tail                                               term                than X + Y                                                                    Tail - 14 days                                                                Occlusion                                                                     time:                                                                         206 = 24 min.                         __________________________________________________________________________     X - Y tail  minimum 14 days in experimental animals                           X + Y tail  minimum 7 days in experimental animals                       

The following additional data was obtained relative to the utility ofthe new compound:

    ______________________________________                                        EFFECT OF NEW COMPOUND ON ELECTRICALLY IN-                                    DUCED THROMBOSIS IN RAT MESENTERIC VESSELS                                    ______________________________________                                        SINGLE LOADING DOSE                                                           20mg/100g B.W.       MALE                                                     MELTING POINT - 147° C.                                                1 DAY AFTER SINGLE LOADING DOSE                                               1 Rat    The occlusion time was 240 minutes                                   3 DAYS AFTER SINGLE LOADING DOSE                                              1 Rat    The occlusion time was 150 minutes                                   2 Rat    The occlusion time was 195 minutes                                   ______________________________________                                    

The following data relates to rat-mensentery occlusion time:

    ______________________________________                                        RAT-MESENTERY OCCLUSION TIME                                                              OCCLUSION                                                                     TIME        CONTROL                                               ______________________________________                                        PABA                                                                          35 mg p.o./d × 3 d (F)                                                                95 min (1F)   45 + 5 min (3F)                                   35 mg p.o./d × 3 d (M)                                                                53 ± 13 min (7M)                                             L-ASCORBIC ACID                                                               10mg/100g body weight/                                                        per day × 3 days (F)                                                                  112 ± 18 min (5F)                                                                        45 ± 5 min (2F)                                10mg/100g body weight/                                                        per day × 3 days (M)                                                                  135 ± 14 min (2M)                                                                        38 ± 10 min (3M)                               PABA +                                                                        L-ASCORBIC ACID                                                               35 mg + 10mg/100g                                                             body weight   108 ± 23.0 min (6F)                                                                      45 ± min (2F)                                                128 ± 18.0 min                                                                           38 ± 10 min (3M)                                             (2M)                                                            10 mg + 10 mg 160 min (2F)  50 minutes (1F)                                   (3M + 3F)     175 min (3M)  55 minutes (1M)                                                 AF + 3M                                                         PABA -                                                                        L-ASCORBIC ACID                                                               SALT                                                                          (NEW COMPOUND)                                                                20 mgs/100 grams                                                                            1 day after Single                                                            Loading Dose                                                                  240 min (1F)                                                                  3 days                                                                        after Single                                                                  Loading Dose                                                                  150 min (1M)                                                                  195 min (2F)                                                    ______________________________________                                    

The following data relates to the occlusion time tail in female rats:

    ______________________________________                                        (X - Y) - NEW COMPOUND - SINGLE DOSE/P.O.                                     100mg/100g body weight                                                                        Average Occluding Time                                        ______________________________________                                        1 day           206 ± 24.0 min (5F)                                         5 days         143 ± 9.5 min (5F)                                          9 days         137 ± 12.0 min (5F)                                        11 days         127 ± 34.0 min (4F)                                        14 days         67 ± 4.5 min (4F)                                                          Control - 48 ± 1.1 min (5F)                                ______________________________________                                    

Below are tabulated some physical characteristics of batches of thecompound which were made:

    ______________________________________                                                MELTING POINT                                                                             SOLUBILITY                                                ______________________________________                                        Batch 1   157° C.                                                                              Sparingly Soluble;                                                            1gm/100cc. H.sub.2 O                                  Batch 2   157° C.                                                                              Same                                                  Batch 3   147° C.                                                                              Same                                                  ______________________________________                                    

Referring next to the sole FIGURE of the drawing, it is seen that thereare illustrated the effects of a single loading dose on a number ofrats. A control is provided in the form of five animals and it is notedfrom the chart in the drawing that the control provides a rat-mesenteryocclusion time relative to electrically-induced thrombosis which is, atthe outset, less than a single loading dose of the new compound after 14days.

More particularly, it will be noted that five animals were sacrificedafter one day following administration of the new compound, five animalswere sacrificed after five days, five more animals were sacrificed afternine days, four additional animals were sacrificed after 11 days andfinally, four animals were sacrificed after 14 days. The occlusion time(in minutes) after the first day is markedly greater than that of thecontrols. After five days, the occlusion time is reduced but is stillmore than double that of the controls. Similarly, after nine days, theocclusion time is substantially greater than the controls and hasreduced only very slightly from the fifth day measurements. Similarly,after 11 days, there is very little reduction in occlusion time which isstill at least twice as great as that of the controls. After 14 days,measurement of four sacrificed animals still reveals an occlusion timewhich is greater than that of the controls.

The measurements in the drawing are based upon an administration of thecompound in a dosage of 100 mg/100 g of body weight and single loadingdoses are employed for both the controls and the animals to which thenew compound has been administered. This shows a substantial tail inuresto the benefit of administration of the new compound and this isimportant with respect to the treatment of humans wherein oraladministration of the new compound is expected to lead to a scheduledadministration which provides for spaced-oral dosages over a period ofdays, such as, for example, one oral administration per week.

The following additional compounds have been made for the above utility:

EXAMPLE III

An antithrombotic compound is prepared in the form of the salt ofglyceryl p-aminobenzoate and ascorbic acid. The structure is: ##STR7##

Method of Preparation

This salt was prepared by dispersing equal molar portions of the twosubstances in a mixture of methanol and acetone. The mixture is heateduntil everything is in true solution. The solvent is then evaporateduntil precipitation begins. At that point the mixture is chilled. Thesalt which crystalizes out is then collected and dried in vacuum. Thecharacteristics of the molecule are as follows:

Melting point--117°-152° C. (broad range probably reflects salt tendencyto decompose rather than give a clean melting point).

The NMR and IR curves of the salt appear to be different from the NMRand IR curves of the individual components, indicating a new molecule ispresent.

EXAMPLE IV

An antithrombotic compound is prepared in the form of the salt of ethylp-aminobenzoate and ascorbic acid. The structure is: ##STR8##

Method of Preparation

The method is identical to that for the ethyl p-aminobenzoate salt.

Melting point--88°-168° C. (broad range reflects salt decomposition).

The IR and NMR analyses show unique spectral differences from thecomponent compounds.

EXAMPLE V

An antithrombotic compound is prepared in the form of esters ofp-aminobenzoic acid with ascorbic acid. A mixture of esters consistingof more than one of the following structures has been prepared. IR andNMR data have been obtained to show the presence of ester bonding. Thinlayer chromatography shows the presence of more than one component. Thestructures for esters are: ##STR9##

Method of Preparation

The esters were prepared by reacting p-nitrobenzoyl chloride withascorbic acid in a solvent with a basic component chloride acceptorpresent, such as pyridine or triethanolamine. By reacting thesematerials under reflux conditions, esterfication occurs with the releaseof hydrochloric acid, as shown in the following schema.

p-nitrobenzoyl chloride+ascorbic acid

mono-ester (s)+HCl

di-ester(s)+HCl

tri-ester+3 HCl

After the isolation by evaporation of the solvents, the nitroesters arereacted with suitable reducing agents, which will only reduce the nitrogroup. These include zinc and hydrochloric acid and lithium aluminumborohydride. Other reducing agents could be used.

The new compounds were isolated by evaporation of the solvent. Removalof all color was accomplished by treatment of the solvent solution withactivated carbon. The product is a mixture of some or all of the estersof p-aminobenzoic acid with ascorbic acid.

There will now be obvious to those skilled in the art, manymodifications and variations of the above methods and compounds. Thesemodifications and variations will not depart from the scope of theinvention if defined by the following claims or if generally equivalentthereto.

What is claimed is:
 1. P-carboglyceryloxy ammonium ascorbate: ##STR10##